Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020

Background The pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens. Aim We aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin. Methods Fifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics. Results Only RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide. Conclusion Some RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.


Introduction
The pet industry has evolved in recent decades due to increasing pet populations, stronger human-pet bonds and demand for high-quality pet food products [1,2].Processed pet food manufactured with various processing methods (e.g.grinding, cooking, extrusion and dehydration) has traditionally been considered microbiological safe and nutritionally suitable for feeding pets [1,3].However, since some pet owners consider unprocessed food healthier, raw meat-based diets (RMBDs) for dogs have gained popularity [1,2,4].The RMBDs are mainly composed of uncooked or minimally processed meat, bones and organs, with freezing as the primary treatment, and are considered to be more natural than conventional processed pet food [1,5].Nevertheless, the scientific evidence supporting RMBD benefits is scarce, and many veterinary professional organisations (e.g. the World Small Animal Veterinary Association) and international public health agencies (e.g. the United States (US) Centers for Disease Control and Prevention (CDC)) view them as potential health hazards for both animals and humans [1,5]; awareness of this issue appears less evident in Europe [6].The safety concerns associated with RMBDs are related to the potential contamination of raw ingredients with zoonotic pathogenic bacteria and parasites [1,3,4].Such contamination could lead to the spread of these pathogens to both pets and humans cohabitating with pets, through direct contact with the pet or its feed, or indirectly through contact with contaminated household surfaces or hands during feed preparation.
In the European Union (EU), legal requirements for the use of animal by-products and derived products not intended for human consumption are established, including those to produce processed or raw pet food, helping to ensure microbiological safety [7].Nevertheless, since 2020, there have been more than 20 notifications or recalls of pet food and RMBD in the EU due to the detection of zoonotic pathogens, particularly Salmonella and pathogenic Escherichia coli [8], and also cases of human infections with Salmonella and Shiga toxin-producing E. coli (STEC) linked to exposure to RMBDs [9][10][11].Several studies have also established a correlation between the microbiota of pets and their owners, including the presence of antibiotic-resistant strains, with pet food as a potential source [12,13].However, certain antibiotic-resistant bacteria and genes of public health concern, such as the mcr gene conferring resistance to the last-line antibiotic colistin, have not been extensively studied in pet food and RMBDs [1,[14][15][16].Consequently, these antibiotic-resistant strains and genes have not been recognised as notable food safety issues in the context of the pet food industry [6].To address this knowledge gap, we aimed to investigate the occurrence of and further characterise Salmonella and other Enterobacteriaceae resistant to critical antibiotics, such as colistin, in dog food, including RMBDs, that is available in stores in Portugal to investigate if they represent a possible source of these hazards to public health.

Sampling strategy
We visited physical locations such as major supermarkets and pet stores in the Porto metropolitan area and conducted an online search to gather information on the primary canine food types and brands commercially accessible in Portugal.Over 5 months (September 2019 to January 2020), 55 dog food samples (22 wet, 14 rawfrozen, eight dry, seven treats and four semi-moist), corresponding to 50 different dog food items (four food types were acquired 2-3 times) and 25 brands commercialised in Portugal, were collected from 12 retail stores (eight supermarkets, three specialised stores and one veterinary clinic) in the Porto region; further details about the samples are appended in Supplementary Table S1.Most samples were obtained from brands marketed globally, including in the EU (21/25).The 14 raw-frozen dog food samples were a combination of fruits, vegetables and different types of meat (muscle/ viscera).They were categorised into two groups based on the main meat type: poultry (n = 8; chicken, turkey, duck, goose) and ruminant (n = 6; veal, steer, deer) samples.The RMBDs were from the two international brands available in Portuguese stores (brand A types produced in the EU and brand B in the United Kingdom (UK)).The samples were processed according to their type, as previously described [17].For the pre-enrichment step at 37 °C for 16-18 h, 25 g of each sample were homogenised (2 min in a Stomacher blender) with 1:10 buffered peptone water (BPW).

What did you want to address in this study and why?
Raw meat-based diets (RMBDs) are increasingly popular among pet owners.However, the potential role of RMBDs has been neglected as a new source of bacteria resistant to last-resort antibiotics which could affect people co-living with pets.We wanted to analyse if dog food from brands sold in the European Union represents a possible source of Salmonella or other bacteria resistant to the important antibiotic colistin.

What have we learned from this study?
Conventionally processed pet food is a safer option than RMBDs.This is because RMBDs of European brands can carry multidrug-resistant bacteria, including globally disseminated pathogenic Salmonella and E. coli harbouring genes encoding resistance to colistin, an antibiotic critically important for human medicine.These hazards are frequent in food-animal production and are causing infections in humans worldwide.

What are the implications of your findings for public health?
The detection in RMBDs of a predominant pandemic Salmonella clone and pathogenic E. coli carrying mobile colistin resistance genes may pose a potential risk of human exposure.This can occur through handling of pet food and/or environmental release by pets.These findings indicate a need for proactive actions involving the pet industry, food safety agencies, and pet owners to mitigate risks for public health.

Detection of non-typhoidal Salmonella
Salmonella detection was performed using the International Standard Organisation [18] method for foodstuffs.Briefly, after the pre-enrichment, 0.1 mL and 1 mL of the BPW were transferred to Rappaport-Vassiliadis medium with Soya (RVS) and Muller-Kauffmann tetrathionate-novobiocin (MKTTn) broths, respectively, for selective enrichment (RVS at 41.5 °C for 24 h and MKTTn at 34-38 °C for 24 h).These broths were then streak-plated on xylose lysine deoxycholate agar and CHROMagar Salmonella Plus.Presumptive Salmonella colonies recovered from both selective agar plates (up to five colonies per plate) were confirmed by biochemical tests (e.g.API-20 E, bioMérieux, Marcy l'Etoile, France), by agglutination with Salmonella O poly antisera and serogroup-specific antisera (Becton Dickinson, New Jersey, US) and by PCR for invA gene detection and Salmonella serotypes of particular concern in the EU (Enteritidis, Typhimurium and 1,4, [5],12:i:-) [19].

Screening of mcr-carrying Enterobacteriaceae
After BPW enrichment, 100 µL and 10 µL were spread on Tryptone Bile X-glucuronide agar plates (TBX) and Simmons citrate agar + inositol (SCAi) with and without colistin (3.5 mg/L) and incubated (TBX at 37 °C for 24 h; SCAi at 37 °C for 48 h) for E. coli and Klebsiella spp.detection, respectively.From each plate, between one and five colonies of each morphotype were spread on a CLED medium for further identification by matrixassisted laser desorption-ionisation-time of flight mass spectrometry (MALDI-TOF VITEK MS, bioMérieux) and standard PCRs for E. coli and K. pneumoniae [20].Colistin resistance genes (mcr-1 to mcr-5 and mcr-6 to mcr-9) were identified in E. coli, K. pneumoniae and S. enterica isolates using a multiplex PCR published previously [21].Amplified simplex PCR products were purified using the NZYGelpure kit (NZYTech, Lisbon, Portugal) and sequenced at Eurofins Genomics (Konstanz, Germany).

Comparative genomic analysis of Salmonella and mcr-1-carrying Escherichia coli
We conducted a comparative genomic analysis using core-genome MLST (cgMLST) between our isolates and genomes queried from Enterobase as well as the hierarchical clustering of cgMLST (HierCC) (https:// enterobase.warwick.ac.uk).These strains were used to develop a minimum-spanning tree using GrapeTree (https://achtman-lab.github.io/GrapeTree/MSTree_holder.html) and MSTreeV2.Metadata of the included Salmonella and E. coli isolates were retrieved from Enterobase (isolate name, cgST, country, year, source).In addition, we conducted a search of antibiotic resistance, metal tolerance and virulence genes as described in the previous section.

Statistical analysis
Occurrence rates and antibiotic-resistant E. coli variations across food types were assessed using Fisher's exact test (α = 0.05).The 95% confidence intervals (CI) for proportions were calculated using Wilson CI.Both analyses were computed using Prism v 9.1.1(GraphPad, Boston, Massachusetts, US).

Detection and characterisation of Salmonella
In our study of 55 pet food samples (41 processed and 14 raw), only raw samples tested positive for Gram-negative bacteria, including the zoonotic pathogen Salmonella, along with bioindicators E. coli and Klebsiella pneumoniae (Table 1).We detected Salmonella in one of the raw samples (7%; 95% CI: 1.3-31.5;n = 1/14), a rawfrozen batch (EU, brand A), predominantly containing turkey (Table 1).

B. Grape tree of E. coli ST297/cgST193137 (HC100-3512) isolates; inset: pet food isolates and related genomes
The core genome minimum spanning tree was created within the EnteroBase pipeline using the MSTreeV2 algorithm and GrapeTree tool.The cgST is indicated in each node.The yellow circle highlights the pet food isolate of each Grape tree.For the geographical analysis, the cgST was annotated using the country data (the number of genomes by country is indicated within parentheses).The scale bar corresponds to the number of cgMLST allelic differences.
Italy, the Netherlands, Ecuador, Kenya and Uganda; 2014-2021), including from a Portuguese poultry farm (Figure 1B); Supplementary Table S3 provides further strain details.The ST3997-ST10 Cplx (cgST193139) presented virulence genes associated with avian pathogenic E. coli (APEC) and shared a single cluster at the HC50 level with five isolates from humans in Europe (Czechia, 2020) and Asia (China, 2017) (Figure 1-C); for further strain details we refer to Supplementary Table S4.Whole genome sequencing revealed that in both E. coli strains from the same pet food brand, the mcr-1.1 gene was located on similar IncX4 plasmids (99.89% identity).These plasmids shared a common genetic environment near the mcr-1 cassette, contained the pap2 gene (membrane-associated lipid phosphatase) and lacked the ISApI1 element [16].
Comparative genomics revealed that these IncX4 plasmids were similar to others (MOB-recon; mash distance: 0.000780658-0.00126265)and are circulating among diverse hosts (humans, pig, poultry) and the environment in many different countries, including Portugal (Figure 2); Supplementary Table S5 provides further plasmid details.

Discussion
This study investigated the presence and characteristics of Salmonella and other Enterobacteriaceae in 55 dog food samples, with a focus on colistinresistant strains.These samples comprised various types of meat and were obtained from different suppliers and international brands in Portugal.We found Enterobacteriaceae, including Salmonella and MDR isolates, only in samples from raw pet food, in contrast to a parallel study in the same samples [17], where Enterococcus spp. was detected across all sample types, including dry and wet.Current regulations in the EU propose counting Enterobacteriaceae (and including Salmonella detection) as a hygiene criterion for all categories of pet foods [7].Numerous studies have demonstrated contamination levels exceeding the  EU limits (i.e.Salmonella: absence in 25 g and Enteroba cteriaceae < 5 × 10 3 CFU/g) in RMBDs [4,5,14,15,27].Our results strongly suggest that conventionally processed pet food is a safer option, emphasising the critical role of heat treatment in pet food production for effectively mitigating microbiological hazards [1,2].
Although the overall prevalence of Salmonella in the RMBDs samples in this study was low (one of 14 batches produced in the EU had unsatisfactory microbiological quality), other studies from Europe also detected Salmonella: 4% of raw pet food samples in Switzerland, 20% in the Netherlands and 71% in Italy [14,16,27].The Salmonella enterica detected in this study was of the serotype 1,4, [5],12:i:-and belonged to ST34, which has emerged as the predominant pandemic genotype in recent decades, particularly in food animal production and human infections in the EU [26,28].Their MDR features (ASSuT + ICE) may have facilitated the adaptation of this serotype to environments with extensive usage of antibiotics and heavy metals, such as pig and poultry farms [26,28,29], whose raw animal by-products are the sources of the pet food industry.Since food animals are asymptomatic carriers of Salmonella, these bacteria can spread easily at slaughterhouses through cross-contamination events between flocks or animal by-products or at pet food production plants, in various types of meat, animal species and geographical places of origin [6,19,30].Notably, we showed genetic similarities between S. 1,4, [5],12:i:-from RMBDs and public genomes from human clinical cases from different European countries, suggesting a role of raw pet food as a potential vehicle for the transmission of this serotype considered of human health significance in the EU and carrying a MDR profile.Some studies consistently show a significant difference in Salmonella excretion in faeces between dogs fed with RMBDs and those fed with dry food, highlighting the microbiological risk associated with RMBDs [3,31,32].This risk extends not only to dogs but also to pet owners handling RMBD and dog faeces, as well as to the environment, as documented by recent Salmonella outbreaks where WGS confirmed a connection between pets, pet food and human disease [10,11,33,34].
A high percentage of our samples carried MDR E. coli isolates, regardless of the raw food types tested, similar to a recent parallel study focused on MDR Enterococcus in dog food in Portugal [14].Resistance to commonly used veterinary antibiotics such as β-lactams, fluoroquinolones, tetracycline and sulphonamides was especially pronounced, mirroring trends seen in other European studies on pet food samples of diverse origins [14,35].The use of these antibiotics, particularly in poultry production, has been associated with increased E. coli resistance rates [29], suggesting that raw meat-based ingredients might be introducing MDR strains in pet food, which then can persist until they reach humans and their pets [32].While the percentage of samples containing MDR and mcr-carrying E. coli isolates was relatively low, in line with findings from other studies [14,16], it underscores the importance of employing antibiotics judiciously within the livestock industry.This is needed to curb the co-selection of genes conferring resistance to colistin, a "highest priority critically important antimicrobial" for human medicine among various bacterial species [20,21].In fact, E. coli ST297 (ExPEC) and ST3997-ST10 Cplx (APEC) identified in this study have been detected worldwide in various animal, food, environmental and human sources, and have been linked to numerous human infections (https:// enterobase.warwick.ac.uk/species/index/ecoli), which highlights their capacity to be transmitted to humans through the food chain.In Portugal, the MDR E. coli ST297 lineage is predominant in many food sources [20,36] and has now been detected in raw pet food in our study.Meanwhile, in Asia, E. coli ST3997-ST10 Cplx isolates found in poultry, the environment and humans also carried mcr-1 associated with diverse plasmid backgrounds [37].Notably, both E. coli strains obtained in this study from the same pet food brand carried the mcr-1.1 gene on similar IncX4 plasmids.These findings, along with the similarity of these plasmids to globally distributed ones, suggest possible crosscontamination events and/or diverse origins of pet food contamination arising from ingredients or human handling at the production plant.
In this study, RMBDs were identified as a potential vehicle of MDR zoonotic-related pathogenic bacteria, with some carrying genes such as mcr-1 conferring resistance to last-line antibiotics.Despite the EU›s efforts to reduce antibiotics such as colistin in livestock production and the successful colistin restrictions on EU farms [20,21], the introduction of colistin-resistant bacteria through imported animal by-products (e.g. from non-EU countries with different antibiotic practices and regulations), raw vegetables (common in most samples) or wildlife (e.g.deer as the main ingredient in one batch with mcr-carrying E. coli) cannot be excluded.Continuous vigilance is essential to address these potential pathways and mitigate the spread of antibiotic-resistant bacteria.Furthermore, most manufacturers do not provide information on food safety practices (e.g.handwashing, safe handling) for handling raw pet food [38], including on labels found on the raw pet food samples obtained for this study.Appropriate hygiene measures and safe handling practices should be observed when dealing with pets and raw pet food to mitigate the risk of MDR bacterial infections in humans.
Finally, we acknowledge the study's limitations.Firstly, the results should be interpreted considering our convenience-based sampling strategy, which exclusively captured dog food types and brands available on the Portuguese pet food market, primarily in four cities in the Porto metropolitan area.Consequently, the results may not be extrapolated to pet food products available from other suppliers.Secondly, the small sample size and the uneven distribution among suppliers and various canine food items may have introduced unintended selection bias.Moreover, additional studies, encompassing brands available in every region in the world, along with local risk assessment investigations, are required to discern the broader implications of pet food on public health.

Conclusion
This study demonstrates that RMBDs from European brands available in Portugal can be a vehicle for MDR clinically-relevant Salmonella and E. coli carrying genes encoding resistance to the last-resort antibiotic colistin.Promoting awareness of potential risks linked to RMBDs and providing guidance to pet owners on proper handling and feeding practices are crucial steps in minimising potential health risks.Identifying environmental transmission routes of pathogenic and MDR bacteria to pet food and the continuous microbiological monitoring (pathogens and antibioticresistant bacteria/genes) of ingredients and processes used in the fast-growing pet food industry needs to be addressed in future One Health studies to mitigate public health risks.

Ethical statement
Ethical approval was not required for this study because the samples were exclusively from dog food acquired in retail stores.

Preprint
A pre-print version of our manuscript, titled "Raw meatbased diet for pets: a neglected source of human exposure to Salmonella and mcr-carrying Escherichia coli in Europe" by Ribeiro-Almeida, Marisa, et al., has been deposited on SSRN, and the corresponding DOI for the preprint is http:// dx.doi.org/10.2139/ssrn.4532439, as posted on 9 August 2023.

License, supplementary material and copyright
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This article is copyright of the authors or their affiliated institutions, 2024.

Figure 2
Figure 2Circular maps of mcr-1-harbouring IncX4 plasmids in Escherichia coli isolated from Portuguese pet food, September 2019-January 2020 (n = 2) and closely related ones from different sources and geographical regions

Table 1
14)tribution of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae among samples of commercially available raw-frozen dog food, Portugal, September 2019-January 2020 (n =14) −: not detected; +: detected.aRawfood samples were from either of two international brands distributed worldwide, here randomly designated as A and B.bThe most prevalent ingredient in each food type is shown in bold.

Table 3
Characteristics of Escherichia coli recovered from samples of commercially available raw-frozen dog food, Portugal, September 2019-January 2020 (n = 59 isolates)